Mammalian chaperonin 10 (Cpn10), also known as heat shock protein 10 (Hsp10) and early pregnancy factor (EPF), is typically characterized as a mitochondrial “molecular chaperone” protein involved in protein folding together with chaperonin 60 (Cpn60; Hsp60). Cpn10 and Cpn60 are homologues of the bacterial proteins GroES and GroEL respectively. GroES and Cpn10 each oligomerize into seven member rings that bind as a lid onto a barrel-like structure comprising fourteen GroEL or seven Cpn60 molecules respectively, which tether denatured proteins to the complex (Bukau and Horwich, 1998, Cell 92:351-366; Hartl and Hayer-Hartl, 2002, Science 295:1852-1858).
Cpn10 proteins are highly conserved across species. Human Cpn10 is 100% identical to bovine Cpn10 and differs from rat Cpn10 at only a single amino acid position. Human Cpn10 shares 30% sequence identity (60% similarity) with GroES from Escherichia coli. As illustrated by the heptameric crystal structure of E. coli GroES (see FIG. 1A; Xu et al., 1997, Nature 388:741-750). Cpn10/GroES proteins are comprised of essentially three different structural regions, an anti-parallel β-barrel region which is flanked by a “roof” β-hairpin loop region and a “mobile loop” region. The mobile loop region mediates interaction with Cpn60/GroEL and is thus critical for the formation of the complex with Cpn60/GroEL and for the “molecular chaperone” protein folding activity.
However in addition to its intracellular role as a molecular chaperone, Cpn10 is also frequently found at the cell surface (see Belles et al., 1999, Infect Immun 67:4191-4200) and in the extracellular fluid (see Shin et al., 2003, J Biol Chem 278:7607-7616) and is increasingly being recognized as a regulator of the immune response. For example, Cpn10 has been demonstrated to have immunosuppressive activity in experimental autoimmune encephalomyelitis, delayed type hypersensitivity and allograft rejection models (Zhang et al., 2003, J Neurol Sci 212:37-46; Morton et al., 2000, Immunol Cell Biol 78:603-607).
The present inventors have also recently demonstrated that Cpn10 can inhibit LPS-induced activation of NF-κB, reduce LPS-induced TNFα and RANTES secretion and enhance IL-10 production in a number of different human and murine in vitro systems and murine disease models (Johnson et al., 2005, J Biol Chem 280:4037-4047 and International Patent Application No. PCT/AU2005/000041, the disclosure of which is incorporated herein by reference), suggesting that Cpn10 has considerable potential as an immuno-therapeutic for the treatment of autoimmune and inflammatory diseases.
However the site(s) within the Cpn10 molecule responsible for mediating this immunomodulatory activity(ies) has remained elusive.
The present invention relates to modifications to the Cpn10 molecules and the effect of these modifications on immunomodulatory activity.